PROJECT SUMMARY Bacterial pathogens and their infectious sequelae remain a huge burden on the health care system, sickening millions in developed countries and killing tens of millions (often children) in developing countries. Many clinically relevant bacterial pathogens establish an intracellular niche in order to replicate, survive and/or persist within the host. These intracellular bacteria either occupy a membrane-bound vacuole or lyse their nascent phagosome to live freely within the cytosol. The fundamental processes governing intracellular niche selection are poorly understood. Here we propose to fill this knowledge gap and investigate whether a key virulence determinant to Gram-negative bacteria, the type III secretion system (T3SS), directs the intracellular lifestyle of pathogenic bacteria. The T3SS or injectisome, is a complex needle-like nanomachine anchored in the bacterial membrane that acts as a conduit for the passage of bacterial effectors directly into the host cell. Contact of the needle tip with host cell membranes, specifically the plasma membrane and endocytic bacteria- containing vacuole membrane, triggers the formation of a membrane-spanning translocon pore. Two bacterial proteins, known as translocators, oligomerize to form this pore. We have found that replacing the gene encoding a translocator protein in the vacuolar bacterium, Salmonella enterica serovar Typhimurium (STm), with its ortholog from a cytosolic bacterium, either Shigella flexneri or Chromobacterium violaceum, allows STm to proficiently lyse its nascent vacuole and colonize the cytosol. Based upon these findings, we hypothesize that intrinsic properties of the translocator proteins define the efficiency of bacteria-containing vacuole lysis, and therefore the intracellular niche occupied by Gram-negative pathogens. We will test our hypothesis by pursuing two Specific Aims. First, we will determine whether translocator proteins from cytosolic bacteria have greater intrinsic membrane-destabilizing activity than those from vacuolar bacteria. Here we will genetically replace translocator proteins in a vacuolar (STm) and cytosolic (C. violaceum) pathogen with orthologs from other members of the Inv/Mxi-Spa T3SS family and measure the effect on bacteria-containing vacuole lysis. We will also construct translocator chimeras to identify functional regions that define their phagolytic properties. Second, we will identify whether the translocon itself or type III effector activities of the translocator proteins account for differential bacteria-containing vacuole lysis. Our proposed studies will specifically address the role of type III translocators in phagosomal membrane lysis. This will improve our understanding of the pathogenic mechanisms utilized by bacteria to colonize host cells, and could aid in the rational design of therapeutics against the T3SS injectisome, which is shared by many clinically relevant Gram- negative pathogens.